Terpenoid Esters Are the Major Constituents From Leaf Lipid Droplets of Camellia sinensis

Abstract

Lipid droplets (LDs) have been widely found from diverse species and exhibit diverse functions. It remains unexplored what potential roles they played in tea. To address this question, we analyzed the chemical composition and the dynamic changes of cytosolic LDs during leaf growth and diurnal cycle. Using TopFluor cholesterol and Nile Red staining we demonstrated that cytosolic LDs were heterogeneous in tea tree (Camellia sinensis cv. Tieguanyin); the size and number of LDs increased with leaf growth. Compositional analysis showed that terpenoid esters and diacylglycerol are the major components of cytosolic LDs. The contents of total sterol esters (SEs) and β-amyrin esters increased with leaf expansion and growth; individual SE also showed diurnal changes. Our data suggest that cytosolic LDs from tea tree leave mainly serve as storage site for free sterols and triterpenoids in the form of esters. Cytosolic LDs were not the major contributors to the aroma quality of made tea.

Keywords: Camellia sinensis; CsAST1; CsHMGRs; CsPSAT1; lipid droplets; neutral lipids; sterol esters; triterpenoid esters.

FIGURE 1

Nile Red staining of leaf lipid droplets from Camellia sinensis cv. Tieguanyin. The third leaf was harvested from a growing twig (A), leaf discs were infiltrated in Nile Red solution (2 μg mL^-1) in 50 mM PIPES buffer (pH 7) for 30 s, then stained for 30 min before observation under confocal microscopy, excitation was at 488 nm and emission was at 550–630 nm (B). Bar = 20 μm.

FIGURE 2

LDs imaging and TLC analysis. Fresh tea tree leaf discs were sequentially stained by TopFluor cholesterol and Nile red, then imaged for TopFluor cholesterol signal (A) and Nile Red signal (B), images (A,B) were overlaid in (C), bar = 10 μm. The LDs stained by Nile Red only were indicated by arrows, the LDs stained by both TopFluor cholesterol and Nile red were indicated triangle; (D) LDs were isolated from tea tree leaves, and stained by TopFluor cholesterol, bar = 20 μm; (E) Total lipid were isolated from purified LDs, then separated on TLC plate. The elution buffer was hexane: diether ether: acetic acid (80:20:1, v/v/v); (F) Total lipids were isolated from fresh tea leaves and analyzed by TLC. The elution buffer was hexane: diether ether: acetic acid (90:7.5:1, v/v/v). SE, sterol ester; TAG, triacylglycerol; FA, fatty acid; 1, 2-DAG, 1, 2-diacylglycerol.

FIGURE 3

Compositional analyses of LDs by GC-MS. (A) GC-MS chromatography of terpenoid esters. Total lipids were isolated from 1.4 g fresh tea leaves, nonpolar lipids were separated by SPE column, then subjected to second SPE column to purify terpenoid esters. The purified terpenoid esters were hydrolyzed by 6% (w/v) KOH in methanol, terpenoids were extracted and derivatized with MSTFA/TMCS for GC-MS analysis. 1, Cholesterol; 2, 1-triacontanol; 3, stigmasterol; 4, β-amyrin; 5, lupeol; 6, β-sitosterol; 7, cycloartenol; 8, campesterol. (B,C) TLC analysis of terpenoid esters isolated from tea leaves without pre-wax removal and with pre-wax removal before total lipid isolation, the elution buffer was hexane: diether ether: acetic acid (80:20:1, v/v/v). Lane 1, β-amyrin standard; lane 2, sterol ester standard; lane 3, total lipids from tea leaves; lane 4, non-polar lipids isolated from total lipids; lane 5, terpenoid esters isolated from non-polar lipids.

FIGURE 4

The contents of sterol esters and triterpenoid esters changed with leaf growth. (A) Sterol ester content changes from the first leaf to the fifth leaf. (B) Triterpenoid ester content changes from the first leaf to the fifth leaf. The first leaf to the fifth leaf was harvested from growing twigs, and pooled together by leaf position. Terpenoid esters were isolated and quantified from each leaf position. Four biological replicates were prepared; the data was expressed as average ± standard deviation based on dry leaf weight.

FIGURE 5

Sterol and sterol ester biosynthesis-related gene expression analyses during leaf maturation from growing shoots. (A–F) Relative gene expression levels of CsHMGR1, CsHMGR2, CsHMGR3, CsHMGR4, CsPSAT1, and CsAST1 from the first leaf to the fifth leaf, respectively. Total RNAs were isolated from the first leaf to the fifth leaf, respectively. Q-PCR was conducted and 2^-ΔΔCt method was used to quantitate gene expression, CsGAPDH was used as reference gene, Three biological replicates were used for each leaf position. The data was expressed as average ± standard deviation. Statistical analysis was performed against the first leaf position, and significant change (p < 0.05) was labeled with asterisk.

FIGURE 6

Sterol esters and triterpenoid esters changed during diurnal cycle. The third leaf from growing twig was harvested and pooled at 4 h interval, beginning at sunrise (5:00 AM). (A) Diurnal changes of leaf sterol ester contents. (B) Diurnal changes of leaf triterpenoid ester contents. Terpenoids esters were isolated and quantified from the third leaf position. Four biological replicates were prepared, and the data was expressed as average ± standard deviation based on dry leaf weight. Statistical analysis was performed against time point at 5:00 AM, and significant change (p < 0.05) was labeled with asterisk.